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Coxsackievirus A16 elicits incomplete autophagy involving the mTOR and ERK pathways.

Shi Y, He X, Zhu G, Tu H, Liu Z, Li W, Han S, Yin J, Peng B, Liu W - PLoS ONE (2015)

Bottom Line: Interestingly, we found that Immunity-related GTPase family M (IRGM) was crucial for the activation of CA16 infection-induced autophagy; in turn, reducing IRGM expression suppressed autophagy.In summary, CA16 can use autophagy to enhance its own replication.These results raise the possibility of targeting the autophagic pathway for the treatment of hand, foot, and mouth disease (HFMD).

View Article: PubMed Central - PubMed

Affiliation: Pathogenic Organism and Infectious Diseases Research Institute, School of Basic Medical Sciences, Wuhan University, Wuhan, 430071, China; Hubei Province Key Laboratory of Allergy and Immunology, Wuhan, 430071, China.

ABSTRACT
Autophagy is an important homeostatic process for the degradation of cytosolic proteins and organelles and has been reported to play an important role in cellular responses to pathogens and virus replication. However, the role of autophagy in Coxsackievirus A16 (CA16) infection and pathogenesis remains unknown. Here, we demonstrated that CA16 infection enhanced autophagosome formation, resulting in increased extracellular virus production. Moreover, expression of CA16 nonstructural proteins 2C and 3C was sufficient to trigger autophagosome accumulation by blocking the fusion of autophagosomes with lysosomes. Interestingly, we found that Immunity-related GTPase family M (IRGM) was crucial for the activation of CA16 infection-induced autophagy; in turn, reducing IRGM expression suppressed autophagy. Expression of viral protein 2C enhanced IRGM promoter activation, thereby increasing IRGM expression and inducing autophagy. CA16 infection inhibited Akt/mTOR signaling and activated extracellular signal-regulated kinase (ERK) signaling, both of which are necessary for autophagy induction. In summary, CA16 can use autophagy to enhance its own replication. These results raise the possibility of targeting the autophagic pathway for the treatment of hand, foot, and mouth disease (HFMD).

No MeSH data available.


Related in: MedlinePlus

IRGM is required for both CA16 infection and 2C/3C-expression induced autophagy.(A) Overexpression of IRGM promotes CA16-mediated LC3 processing. HeLa cells were transfected with empty vector or HA-IRGM for 24 h, followed by infection with CA16 (MOI = 2). At 12 h after infection with CA16, the cells were subjected to Western blotting using anti-LC3B, IRGM and Vp1 antibodies. (B) Overexpression of IRGM promotes 2C-mediated LC3 processing. HeLa cells were cotransfected with HA-IRGM plus vector or HA-2C. At 24 h after transfection, the cells were subjected to Western blotting using anti-LC3B, IRGM and HA antibodies. (C) Overexpression of IRGM promotes 3C-mediated LC3 processing. HeLa cells were cotransfected with HA-IRGM plus vector or HA-3C. At 24 h after transfection, the cells were subjected to Western blotting using anti-LC3B, IRGM and HA antibodies. (D) Depletion of IRGM expression attenuates CA16-mediated LC3 processing. HeLa cells were transfected with siIRGM or control siRNA for 48 h, followed by infection with CA16 (MOI = 2). At 12 h after infection with CA16, the cells were subjected to Western blotting using anti-LC3B, IRGM and Vp1 antibodies. (E) Depletion of IRGM expression attenuates 2C-mediated LC3 processing. HeLa cells were transfected with siIRGM or control siRNA for 12 h, followed by transfection with vector or HA-2C. At 24 h after the second transfection, the cells were subjected to Western blotting using anti-LC3B, IRGM and HA antibodies. (F) Depletion of IRGM expression attenuates 3C-mediated LC3 processing. HeLa cells were transfected with siIRGM or control siRNA for 12 h, followed by transfection with vector or HA-3C. At 24 h after the second transfection, the cells were subjected to Western blotting using anti-LC3B, IRGM and HA antibodies. β-Actin was used as a protein loading control. Representative results are shown, with graphs representing the ratio of LC3-II to β-Actin normalized to the control condition. Data are presented as the means from three independent experiments. Significance was analyzed with a two-tailed Student’s t test. *P< 0.05, **P< 0.01, ***P< 0.001.
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pone.0122109.g006: IRGM is required for both CA16 infection and 2C/3C-expression induced autophagy.(A) Overexpression of IRGM promotes CA16-mediated LC3 processing. HeLa cells were transfected with empty vector or HA-IRGM for 24 h, followed by infection with CA16 (MOI = 2). At 12 h after infection with CA16, the cells were subjected to Western blotting using anti-LC3B, IRGM and Vp1 antibodies. (B) Overexpression of IRGM promotes 2C-mediated LC3 processing. HeLa cells were cotransfected with HA-IRGM plus vector or HA-2C. At 24 h after transfection, the cells were subjected to Western blotting using anti-LC3B, IRGM and HA antibodies. (C) Overexpression of IRGM promotes 3C-mediated LC3 processing. HeLa cells were cotransfected with HA-IRGM plus vector or HA-3C. At 24 h after transfection, the cells were subjected to Western blotting using anti-LC3B, IRGM and HA antibodies. (D) Depletion of IRGM expression attenuates CA16-mediated LC3 processing. HeLa cells were transfected with siIRGM or control siRNA for 48 h, followed by infection with CA16 (MOI = 2). At 12 h after infection with CA16, the cells were subjected to Western blotting using anti-LC3B, IRGM and Vp1 antibodies. (E) Depletion of IRGM expression attenuates 2C-mediated LC3 processing. HeLa cells were transfected with siIRGM or control siRNA for 12 h, followed by transfection with vector or HA-2C. At 24 h after the second transfection, the cells were subjected to Western blotting using anti-LC3B, IRGM and HA antibodies. (F) Depletion of IRGM expression attenuates 3C-mediated LC3 processing. HeLa cells were transfected with siIRGM or control siRNA for 12 h, followed by transfection with vector or HA-3C. At 24 h after the second transfection, the cells were subjected to Western blotting using anti-LC3B, IRGM and HA antibodies. β-Actin was used as a protein loading control. Representative results are shown, with graphs representing the ratio of LC3-II to β-Actin normalized to the control condition. Data are presented as the means from three independent experiments. Significance was analyzed with a two-tailed Student’s t test. *P< 0.05, **P< 0.01, ***P< 0.001.

Mentions: To further investigate the role of IRGM in autophagy induction, HeLa cells were transiently transfected with HA-IRGM or siIRGM to over-express or knockdown IRGM, respectively. As depicted in Fig 6A, 6B and 6C, the IRGM protein was successfully over-expressed in HeLa cells as detected by Western blotting analysis. Interestingly, there was an obvious increase in LC3-II levels in CA16-infected cells over-expressing IRGM (Fig 6A, P< 0.01). Similarly, LC3-II levels were also evidently increased in both 2C and 3C over-expressing cells (Fig 6B and 6C, P< 0.05) over-expressing IRGM. Next, we depleted IRGM expression using small interfering RNAs (siRNA), resulting in a marked knockdown of IRGM, as confirmed by Western blotting (Fig 6D, 6E and 6F). As shown in Fig 6D, 6E and 6F, there was a prominent decrease in LC3-II conversion in CA16-infected (P< 0.001) and 2C (P< 0.01) or 3C (P< 0.01) over-expressing cells after IRGM inhibition compared to the control. Taken together, these results indicate that IRGM is required for both CA16 infection and 2C or 3C over-expression-induced autophagy.


Coxsackievirus A16 elicits incomplete autophagy involving the mTOR and ERK pathways.

Shi Y, He X, Zhu G, Tu H, Liu Z, Li W, Han S, Yin J, Peng B, Liu W - PLoS ONE (2015)

IRGM is required for both CA16 infection and 2C/3C-expression induced autophagy.(A) Overexpression of IRGM promotes CA16-mediated LC3 processing. HeLa cells were transfected with empty vector or HA-IRGM for 24 h, followed by infection with CA16 (MOI = 2). At 12 h after infection with CA16, the cells were subjected to Western blotting using anti-LC3B, IRGM and Vp1 antibodies. (B) Overexpression of IRGM promotes 2C-mediated LC3 processing. HeLa cells were cotransfected with HA-IRGM plus vector or HA-2C. At 24 h after transfection, the cells were subjected to Western blotting using anti-LC3B, IRGM and HA antibodies. (C) Overexpression of IRGM promotes 3C-mediated LC3 processing. HeLa cells were cotransfected with HA-IRGM plus vector or HA-3C. At 24 h after transfection, the cells were subjected to Western blotting using anti-LC3B, IRGM and HA antibodies. (D) Depletion of IRGM expression attenuates CA16-mediated LC3 processing. HeLa cells were transfected with siIRGM or control siRNA for 48 h, followed by infection with CA16 (MOI = 2). At 12 h after infection with CA16, the cells were subjected to Western blotting using anti-LC3B, IRGM and Vp1 antibodies. (E) Depletion of IRGM expression attenuates 2C-mediated LC3 processing. HeLa cells were transfected with siIRGM or control siRNA for 12 h, followed by transfection with vector or HA-2C. At 24 h after the second transfection, the cells were subjected to Western blotting using anti-LC3B, IRGM and HA antibodies. (F) Depletion of IRGM expression attenuates 3C-mediated LC3 processing. HeLa cells were transfected with siIRGM or control siRNA for 12 h, followed by transfection with vector or HA-3C. At 24 h after the second transfection, the cells were subjected to Western blotting using anti-LC3B, IRGM and HA antibodies. β-Actin was used as a protein loading control. Representative results are shown, with graphs representing the ratio of LC3-II to β-Actin normalized to the control condition. Data are presented as the means from three independent experiments. Significance was analyzed with a two-tailed Student’s t test. *P< 0.05, **P< 0.01, ***P< 0.001.
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pone.0122109.g006: IRGM is required for both CA16 infection and 2C/3C-expression induced autophagy.(A) Overexpression of IRGM promotes CA16-mediated LC3 processing. HeLa cells were transfected with empty vector or HA-IRGM for 24 h, followed by infection with CA16 (MOI = 2). At 12 h after infection with CA16, the cells were subjected to Western blotting using anti-LC3B, IRGM and Vp1 antibodies. (B) Overexpression of IRGM promotes 2C-mediated LC3 processing. HeLa cells were cotransfected with HA-IRGM plus vector or HA-2C. At 24 h after transfection, the cells were subjected to Western blotting using anti-LC3B, IRGM and HA antibodies. (C) Overexpression of IRGM promotes 3C-mediated LC3 processing. HeLa cells were cotransfected with HA-IRGM plus vector or HA-3C. At 24 h after transfection, the cells were subjected to Western blotting using anti-LC3B, IRGM and HA antibodies. (D) Depletion of IRGM expression attenuates CA16-mediated LC3 processing. HeLa cells were transfected with siIRGM or control siRNA for 48 h, followed by infection with CA16 (MOI = 2). At 12 h after infection with CA16, the cells were subjected to Western blotting using anti-LC3B, IRGM and Vp1 antibodies. (E) Depletion of IRGM expression attenuates 2C-mediated LC3 processing. HeLa cells were transfected with siIRGM or control siRNA for 12 h, followed by transfection with vector or HA-2C. At 24 h after the second transfection, the cells were subjected to Western blotting using anti-LC3B, IRGM and HA antibodies. (F) Depletion of IRGM expression attenuates 3C-mediated LC3 processing. HeLa cells were transfected with siIRGM or control siRNA for 12 h, followed by transfection with vector or HA-3C. At 24 h after the second transfection, the cells were subjected to Western blotting using anti-LC3B, IRGM and HA antibodies. β-Actin was used as a protein loading control. Representative results are shown, with graphs representing the ratio of LC3-II to β-Actin normalized to the control condition. Data are presented as the means from three independent experiments. Significance was analyzed with a two-tailed Student’s t test. *P< 0.05, **P< 0.01, ***P< 0.001.
Mentions: To further investigate the role of IRGM in autophagy induction, HeLa cells were transiently transfected with HA-IRGM or siIRGM to over-express or knockdown IRGM, respectively. As depicted in Fig 6A, 6B and 6C, the IRGM protein was successfully over-expressed in HeLa cells as detected by Western blotting analysis. Interestingly, there was an obvious increase in LC3-II levels in CA16-infected cells over-expressing IRGM (Fig 6A, P< 0.01). Similarly, LC3-II levels were also evidently increased in both 2C and 3C over-expressing cells (Fig 6B and 6C, P< 0.05) over-expressing IRGM. Next, we depleted IRGM expression using small interfering RNAs (siRNA), resulting in a marked knockdown of IRGM, as confirmed by Western blotting (Fig 6D, 6E and 6F). As shown in Fig 6D, 6E and 6F, there was a prominent decrease in LC3-II conversion in CA16-infected (P< 0.001) and 2C (P< 0.01) or 3C (P< 0.01) over-expressing cells after IRGM inhibition compared to the control. Taken together, these results indicate that IRGM is required for both CA16 infection and 2C or 3C over-expression-induced autophagy.

Bottom Line: Interestingly, we found that Immunity-related GTPase family M (IRGM) was crucial for the activation of CA16 infection-induced autophagy; in turn, reducing IRGM expression suppressed autophagy.In summary, CA16 can use autophagy to enhance its own replication.These results raise the possibility of targeting the autophagic pathway for the treatment of hand, foot, and mouth disease (HFMD).

View Article: PubMed Central - PubMed

Affiliation: Pathogenic Organism and Infectious Diseases Research Institute, School of Basic Medical Sciences, Wuhan University, Wuhan, 430071, China; Hubei Province Key Laboratory of Allergy and Immunology, Wuhan, 430071, China.

ABSTRACT
Autophagy is an important homeostatic process for the degradation of cytosolic proteins and organelles and has been reported to play an important role in cellular responses to pathogens and virus replication. However, the role of autophagy in Coxsackievirus A16 (CA16) infection and pathogenesis remains unknown. Here, we demonstrated that CA16 infection enhanced autophagosome formation, resulting in increased extracellular virus production. Moreover, expression of CA16 nonstructural proteins 2C and 3C was sufficient to trigger autophagosome accumulation by blocking the fusion of autophagosomes with lysosomes. Interestingly, we found that Immunity-related GTPase family M (IRGM) was crucial for the activation of CA16 infection-induced autophagy; in turn, reducing IRGM expression suppressed autophagy. Expression of viral protein 2C enhanced IRGM promoter activation, thereby increasing IRGM expression and inducing autophagy. CA16 infection inhibited Akt/mTOR signaling and activated extracellular signal-regulated kinase (ERK) signaling, both of which are necessary for autophagy induction. In summary, CA16 can use autophagy to enhance its own replication. These results raise the possibility of targeting the autophagic pathway for the treatment of hand, foot, and mouth disease (HFMD).

No MeSH data available.


Related in: MedlinePlus